Low affinity antibodies are valuable for therapeutic use, such as combination therapies. Low affinity antibodies are also useful in drug discovery. For example, where high affinity antibodies are difficult to obtain for a specific therapeutic molecule, low affinity antibodies can serve as a starting point for developing useful affinity-matured antibodies.
Anti-therapeutic molecule antibody assays, needed for regulatory approval of therapeutic molecules, require low affinity antibodies. Such assays require sensitive and efficient means to detect unwanted immune responses, important for assessing safety and efficacy of a therapeutic molecule.
Anti-therapeutic molecule antibodies can target different regions of the therapeutic and can exhibit differing binding affinities and isotypes. A panel of varied anti-therapeutic molecule antibodies mimicking the polyclonal nature of an immune response is desirable, to more accurately assess performance of anti-therapeutic molecule antibody assays.
Screening hybridoma clones for high affinity antibodies has traditionally utilized ELISA technology. ELISA, however, is not as effective for screening low affinity antibodies. Although ELISA can identify antibodies that bind an antigen, the assay cannot readily identify antibodies that bind with low affinity. Many low affinity antibodies are lost in the multiple wash steps required to ensure a high signal-to-noise ratio. Minimizing wash steps to retain these low affinity antibodies, however, decreases sensitivity of the assay by decreasing the signal-to-noise ratio.
A minimal number of wash steps are required in electrochemiluminescence assay (ECLA), permitting the ECLA system to detect low affinity antibodies that would be washed away by traditional ELISA methods. Simply replacing ELISA with ECLA is not a good solution, however. Like ELISA, ECLA cannot readily identify antibodies that bind with low affinity. In addition, labeling agents used in ECLA have the potential to alter binding properties of the antibodies. ECLA can thus fail to retain antibodies that would otherwise be retained by conventional ELISA methods.
Efficient assay systems and methods are greatly needed for screening a pool of analyte molecules, such as antibodies, to identify those having specific characteristics, including low affinity antibodies, anti-therapeutic molecule antibodies responsive to a variety of epitopes, and the like. In particular, efficient and reliable methods to identify a pool of analyte molecules enriched with those having a desired affinity (low or high) or likely to contain analyte molecules responsive to differing binding sites of a target molecule, would be very useful.